ABSTRACT
BACKGROUND: Migrant mothers in high-income countries often encounter more complications during pregnancy, delivery, and the postpartum period. To enlighten health care providers concerning potential barriers, the objective of this study was to explore positive and negative experiences with maternal health services in the University Hospitals of Geneva and Zurich and to describe barriers to maternity services from a qualitative perspective. METHODS: In this qualitative study, six focus groups (FGs) were conducted involving 33 women aged 21 to 40 years. All FG discussions were audio-recorded and later transcribed. Data were analysed using a thematic analysis approach assisted by the Atlas.ti qualitative data management software. RESULTS: Positive experiences included not only the availability of maternity services, especially during emergency situations and the postpartum period, but also the availability of specific maternity services for undocumented migrants in Geneva. Negative experiences were classified into either personal or structural barriers. On the personal level, the main barriers were a lack of social support and a lack of health literacy, whereas the main themes on the structural level were language barriers and a lack of information. CONCLUSION: Structural adaptation is necessary to meet the needs of the extremely diverse population. The needs include (1) the provision of specific information for migrant women in multiple languages, (2) the availability of trained interpreters who are easily accessible to health care providers, (3) specifically trained nurses or social assistance providers to guide migrants through the health system, and (4) a cultural competence-training programme for health care providers.
Subject(s)
Communication Barriers , Health Knowledge, Attitudes, Practice , Health Personnel/standards , Maternal Health Services/standards , Mothers/psychology , Parturition/psychology , Transients and Migrants/psychology , Adult , Cultural Competency , Female , Focus Groups , Health Services Accessibility , Humans , Maternal Health Services/organization & administration , Postpartum Period , Pregnancy , Qualitative Research , Switzerland , Young AdultSubject(s)
Abdominal Abscess/microbiology , Adnexal Diseases/microbiology , Bacteremia/microbiology , Infant, Premature, Diseases/microbiology , Pneumococcal Infections/microbiology , Puerperal Infection/microbiology , Streptococcus pneumoniae/isolation & purification , Abdominal Abscess/diagnosis , Abdominal Abscess/therapy , Adnexal Diseases/diagnosis , Adnexal Diseases/therapy , Adult , Female , Humans , Infant, Newborn , Infant, Premature , Male , Pneumococcal Infections/congenital , Postpartum Period , Pregnancy , Puerperal Infection/diagnosis , Puerperal Infection/therapyABSTRACT
Genital prolapse is frequent and can be found in about 50% of parous women. Its etiology is complex and multifactorial. Predisposing factors include: genetics (connective tissue disorders, family history); general state (age, parity, weight, smoking, obstructive pulmonary disease); trauma (carrying heavy loads, intense physical exercise); or iatrogenic (post hysterectomy). Treatment can be conservative or surgical and depends mainly on the severity of symptoms. Developments in surgical techniques and synthetic material in the last 20 years enabled us to use minimally invasive procedures with improved post operative course and decreased recurrence rates.
Subject(s)
Laparoscopy , Surgical Mesh , Uterine Prolapse/surgery , Female , Follow-Up Studies , Humans , Middle Aged , Postoperative ComplicationsABSTRACT
OBJECTIVES: To evaluate the technique of laparoscopic lateral colpo-uterine suspension using a mesh to treat pelvic organ prolapse, with a sufficient follow-up. PATIENTS AND METHODS: The technique consists of two steps. First, the lateral suspension of the vaginal vault and of the uterus is performed using a polypropylene mesh placed in the vesicovaginal septum as a transversal hammock. The second step is the application of a polypropylene patch to the posterior surface of the vagina and the rectovaginal septum. The transversal hammock is placed laterally by the tension-free fixation of the mesh to the lateral abdominal wall above the iliac crests. Between January 2004 and December 2007, 218 patients were treated. It is a continuous series including all the patients needing a surgical procedure to treat a genital prolapse. We excluded, from the study, the patients with a severe cardiorespiratory disease and the cases of isolated rectocele. RESULTS: We observed a recurrence of the prolapse in thirty patients (13.76%). A reoperation was performed in 10 patients (4.6%). One complication was related to the technique of lateral suspension (bladder injury immediately sutured 0.46%). A mesh erosion was noted in 13 cases (5.96%), nine were treated by vaginal excision of the mesh (4.12%). CONCLUSIONS: The laparoscopic lateral colpo-uterine suspension using a mesh corrects the pelvic organ prolapse with a predominant cystocele or rectocele. It is an interesting alternative to the other procedures because of the low risk of complications and the satisfactory results.
Subject(s)
Laparoscopy/methods , Pelvic Organ Prolapse/surgery , Surgical Mesh , Aged , Female , Humans , Intraoperative Complications/epidemiology , Laparoscopy/adverse effects , Laparoscopy/instrumentation , Middle Aged , Postoperative Complications/epidemiology , Recurrence , Treatment Outcome , Urinary Incontinence/surgery , Uterus , VaginaABSTRACT
The leading region of the conjugal bacterial plasmid ColIb-P9 contains three dispersed repeats of a 328 bp sequence homologous to Frpo, a sequence from plasmid F that acts as a promoter in single-stranded DNA. One of these sequences, ssi3, inactive in the double-stranded form, promoted in vitro transcription exclusively from the single strand that is transferred during conjugation. Promoter activity was dependent on the presence of RNA polymerase holoenzyme containing sigma 70. Transcription initiated from the position predicted from folding the single-stranded DNA to form a pseudo double-stranded hairpin structure containing recognizable -35 and -10 promoter elements. Footprinting of RNA polymerase holoenzyme on single-stranded ssi3 DNA was consistent with this suggestion. Mutagenesis of the putative -35 region inactivated the promoter, but random mutations in the -10 region had little effect. The putative -10 region is a poor match to the consensus sequence and contains mismatched bases. Elimination of these mismatches invariably destroyed single-strand promoter activity. These observations reveal the crucial contribution of the unpaired bases in the -10 region in potentiating the formation of the productive open complex with RNA polymerase.
Subject(s)
Bacteriocin Plasmids/genetics , DNA, Single-Stranded/physiology , Nucleic Acid Conformation , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid , Transcription, Genetic , Base Pair Mismatch , Conjugation, Genetic , Consensus Sequence , DNA Footprinting , DNA, Bacterial/genetics , DNA, Bacterial/physiology , DNA, Single-Stranded/genetics , DNA-Directed RNA Polymerases/metabolism , F Factor/genetics , Gene Expression Regulation, Bacterial , Mutation , Sequence Homology , Sigma Factor/metabolismABSTRACT
Activation of the lectin pathway of complement is initiated by the binding to microbial carbohydrate structures of a multimolecular fluid-phase complex composed of a carbohydrate recognition subcomponent that associates with three specific serine proteases and an enzymatically inert protein of 19 kDa. The first carbohydrate recognition subcomponent of the lectin pathway identified was mannan-binding lectin (MBL), hence the serine proteases were named MBL-associated serine proteases (MASPs) and numbered according to the sequence of their discovery. Here we describe the primary structures of the two distinct serine proteases MASP-1 and MASP-3 in the rat (and of MASP-3 in the mouse), show their association with plasma MBL complexes, and demonstrate that in rat and mouse, as in man, MASP-1 and MASP-3 are encoded by a single structural gene. For both species, we present the genomic region and regulatory elements responsible for the processing of either MASP-1 or MASP-3 mRNA by alternative splicing/alternative polyadenylation. Furthermore, we demonstrate the evolutionary conservation of MASP-3 mRNA in cDNA transcripts from guinea pig, rabbit, pufferfish, and cow.
Subject(s)
Complement Pathway, Mannose-Binding Lectin/genetics , Mice/genetics , Rats/genetics , Serine Endopeptidases/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Conserved Sequence/genetics , DNA Primers , DNA, Complementary/genetics , Mannose-Binding Protein-Associated Serine Proteases , Molecular Probe Techniques , Molecular Sequence Data , Polyadenylation/genetics , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Serine Endopeptidases/metabolismABSTRACT
Current methods for measuring the efficiency of splicing in mammalian cells rely on either direct analysis of the RNA, which does not lend itself to rapid assays, or on single reporter functions that are subject to numerous intrinsic variables. If two protein activities are encoded within a single reading frame but on separate exons, with an intervening sequence containing termination codons, then the expression of the second activity is dependent on removal of the intervening sequence by pre-mRNA splicing. Thus, the ratio of the activities encoded by exon 2 to exon 1 reflects the ratio of expression from spliced mRNA to the total expression of spliced and unspliced RNA. This provides a rapid and convenient assay for the effects on splicing efficiency of trans-acting factors or of alterations in the sequences of the intron and surrounding exon sequences.
Subject(s)
Genes, Reporter , Genetic Techniques , RNA Splicing , RNA, Messenger/analysis , Animals , Cell Line , Humans , Luciferases/analysis , Luciferases/genetics , Mammals , RNA, Messenger/metabolism , Recombinant Fusion Proteins/analysis , beta-Galactosidase/analysis , beta-Galactosidase/geneticsABSTRACT
The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins/metabolism , RNA Splice Sites , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Alternative Splicing , Binding Sites , Binding, Competitive , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Models, Biological , Nuclear Proteins/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoproteins/genetics , Serine-Arginine Splicing FactorsABSTRACT
The gene encoding heterogeneous ribonucleoprotein (hnRNP) G recently has been mapped to the X chromosome. All mammals have a Y chromosome-encoded homologue of HNRNP G called RBMY, which is implicated with a role in male fertility and is a candidate for the azoospermia factor gene. We have identified a new member of this gene family, HNRNP G-T, and have mapped it as a single-copy gene on chromosome 11. This gene contains an uninterrupted open reading frame and no introns, consistent with derivation from a retroposon. However, unlike many retroposon-derived genes, HNRNP G-T is not a pseudogene. An antiserum raised to the conceptual reading frame of HNRNP G-T showed that it encodes a protein that is highly expressed in germ cells and in particular in the nuclei of meiotic spermatocytes. Surprisingly, although this antiserum was raised against human hnRNP G-T protein, it can also detect a similar protein in the testis of several mammals. This suggests that the protein is highly conserved and that the retrotransposition event generating the HNRNP G-T gene pre-dated at least the common ancestor of mouse and man. The existence of an additional testis-specific hnRNP G family member provides evidence for the importance of these proteins in normal germ cell development.
Subject(s)
Evolution, Molecular , Retroelements/genetics , Ribonucleoproteins/biosynthesis , Ribonucleoproteins/genetics , Spermatocytes/metabolism , X Chromosome , Amino Acid Sequence , Animals , Blotting, Southern , Cattle , Cell Nucleus , Chromosomes, Human, Pair 11 , Cloning, Molecular , Conserved Sequence , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Library , Genetic Linkage , Heterogeneous-Nuclear Ribonucleoproteins , Humans , In Situ Hybridization, Fluorescence , Introns , Male , Meiosis/genetics , Mice , Molecular Sequence Data , Open Reading Frames , Physical Chromosome Mapping , Rats , Sequence Homology, Amino Acid , Testis/metabolism , Two-Hybrid System TechniquesABSTRACT
The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis-specific relative, termed hnRNP G-T, interact with Tra2beta, an activator of pre-mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2beta proteins are associated in HeLa nuclear extracts. RBM and Tra2beta co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2beta. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2beta-dependent splicing in spermatocytes.
Subject(s)
Drosophila Proteins , RNA Splicing , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Spermatogenesis , Y Chromosome , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Male , Nuclear Proteins , Testis/metabolism , Two-Hybrid System TechniquesABSTRACT
Alternative pre-mRNA splicing of two terminal exons (alpha and beta) regulates the expression of the human DNA ligase III gene. In most tissues, the alpha exon is expressed. In testes and during spermatogenesis, the beta exon is used instead. The alpha exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the beta exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the beta exon and extending into the downstream intron derepressed splicing to the beta exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the beta exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the poly-adenylation signal was deleted or replaced by a 5' splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceo-somal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation.
Subject(s)
DNA Ligases/genetics , Exons , Gene Silencing , RNA Splicing , Testis/enzymology , Base Sequence , Cell Nucleus/enzymology , DNA Ligase ATP , DNA Primers , HeLa Cells , Humans , Male , Mutagenesis , Poly-ADP-Ribose Binding Proteins , Xenopus ProteinsABSTRACT
RNA-binding proteins are essential for spermatogenesis: they are required in the nucleus of germ cells, for the production of specific mRNA isoforms, and in the cytoplasm - where proteins required for chromatin condensation and for changes in cell morphology are translated long after transcription ceases. Some of the RNA targets and the RNA-binding proteins themselves have been identified recently. Both nuclear and cytoplasmic proteins are affected in examples of azoospermia in men.
Subject(s)
Infertility, Male/physiopathology , RNA-Binding Proteins/physiology , Spermatogenesis/physiology , Animals , Cytoplasm/metabolism , Humans , Male , Nuclear Proteins/physiologyABSTRACT
RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells. Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown. Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function. The mouse homologue was also cloned and designated étoile. It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8. T-STAR/étoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/étoile and SAM68 is regulated during meiosis. Transfection of T-STAR/étoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment. RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68.
Subject(s)
RNA-Binding Proteins/genetics , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Blotting, Northern , COS Cells , Cell Nucleus/chemistry , Chromosome Mapping , Chromosomes, Human, Pair 8/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA-Binding Proteins , Gene Expression , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Muridae , Plasmids , Protein Binding , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Rats , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Spermatogenesis , Tissue Distribution , Tumor Suppressor Protein p53/geneticsABSTRACT
The production of different transcripts (transcript heterogeneity) is a feature of many genes that may result in phenotypic variation. Several mechanisms, that occur at both the DNA and RNA level have been shown to contribute to this transcript heterogeneity in mammals, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in linear, contiguous DNA or RNA. Here we describe tissue-specific repetition of selective exons in transcripts of a rat gene (SA) with a normal exon-intron organization. We conclude that nonlinear mRNA processing can generate tissue-specific transcripts.
Subject(s)
Exons/genetics , Proteins/genetics , RNA, Messenger/genetics , Repetitive Sequences, Nucleic Acid/genetics , Animals , Cells, Cultured , Coenzyme A Ligases , Kidney Tubules, Proximal/metabolism , Liver/metabolism , Polymerase Chain Reaction , RNA Processing, Post-Transcriptional/genetics , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Restriction Mapping , Ribonuclease H/metabolismABSTRACT
Efficient splicing of the 5'-most intron of pre-mRNA requires a 5' m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP binding to 5' splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5' cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5' splice site and not with any loss of U1 snRNP binding.
Subject(s)
RNA Caps/genetics , RNA Precursors/genetics , RNA Splicing/genetics , RNA, Small Nuclear/genetics , Ribonucleoprotein, U1 Small Nuclear/genetics , Alternative Splicing/genetics , Cell Nucleus/genetics , Cross-Linking Reagents/metabolism , Dinucleoside Phosphates/genetics , Ficusin/metabolism , HeLa Cells , Humans , Kinetics , Ribonuclease H/metabolism , Transcription, Genetic/geneticsABSTRACT
RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.
Subject(s)
RNA Precursors/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Repressor Proteins , Spermatogenesis , Spermatozoa/ultrastructure , Cyclic AMP Response Element Modulator , DNA-Binding Proteins/biosynthesis , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Male , Meiosis , Nuclear Proteins , Prostate/chemistry , RNA/metabolism , Ribonucleoproteins/biosynthesis , Sp1 Transcription Factor/biosynthesis , Spermatozoa/metabolism , Testis/chemistryABSTRACT
Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.
Subject(s)
Dystrophin/genetics , Muscle, Skeletal/metabolism , Oligonucleotides, Antisense/pharmacology , RNA Splicing/drug effects , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Dystrophin/biosynthesis , Exons/genetics , Gene Expression/drug effects , Introns/genetics , Mice , Mice, Inbred mdx , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Point Mutation , Polyethyleneimine/pharmacology , Sarcolemma/metabolism , TransfectionABSTRACT
Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.
Subject(s)
Psoriasis/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Adult , Biopsy , Clone Cells , Female , Humans , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/blood , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Male , Middle Aged , Psoriasis/blood , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/immunology , Reproducibility of Results , Skin/pathology , T-Lymphocytes/cytologyABSTRACT
The effects of DNA gyrase and quinolone drugs on in vitro transcription of a template containing a preferred gyrase cleavage site have been investigated. We have found that gyrase-quinolone complexes with DNA lead to blocking of transcription by Escherichia coli and bacteriophage T7 RNA polymerases. Either gyrase or quinolone alone has no effect on transcription. With DNA gyrase containing a point mutation in the gyrase A protein, known to confer quinolone resistance, blocking was found to occur only at much higher concentrations of the drug. Other agents that inhibit gyrase-catalysed supercoiling (novobiocin and 5'-adenylyl-beta,gamma-imidodiphosphate) do not arrest transcription in the presence of gyrase. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Analysis of transcription in the absence of drug suggests that RNA polymerase does not displace gyrase from the template. These results are discussed in the light of models for the bactericidal effects of quinolone drugs.